Flash photolysis is a technique utilized to study oxidation-reduction reactions in tissues, based on the fluorescence characteristics exhibited by the compounds in the specimen under study. In this technique a short pulse or flash of light is focussed onto a position in a sample; oxidation inhibited compounds are photolysized by the flash of light and are oxidized with a concurrent fluorescence emission. Photoactivatable caged compounds that are biologically inactive until exposed to ultraviolet (UV) light can be activated by flash photolysis. Flash photolysis techniques can be utilized in confocal laser scanning microscopes to provide both imaging and local photolysis of caged compounds. See Samuel S. H. Wang, George J. Augustine, "Confocal Imaging and Local Photolysis of Caged Compounds: Dual Probes of Synaptic Function," Neuron, Vol. 15, October, 1995, pp. 755-760. The confocal epifluorescence microscope can then be utilized to provide an image of the fluorescence emitted from the specimen in response to the pulse of UV light focussed onto a small area in the specimen.
A difficulty encountered in the use of flash photolysis in conjunction with a confocal microscope is the problem of precisely predetermining the position on a specimen at which the pulse of excitation light will be focussed. To change the target position for the excitation light from one position to another on the specimen within the field of view of the confocal microscope, the specimen slide may be moved by the operator to a position at which the excitation light will be focussed on the new target position. However, moving the specimen is generally not desirable for stability reasons. The microscope and/or flash photolysis equipment may also be adjusted based on the operator's estimate of the target position at which the excitation light will be focussed. This estimated position may not correspond exactly to the actual position of incidence of the excitation light on the specimen. In addition, typical microscope objective lenses have a somewhat different index of refraction for the UV light used for flash photolysis excitation than for visible light wavelengths, so that the actual position of incidence as estimated utilizing visible light scanning may not exactly correspond to the position at which the UV excitation light will be focussed by the objective.